Escherichia coli selD gene was mutated by site-specific mutagenesis. The selD gene product, SELD protein, catalyzes the ATP-dependent formation of a diffusible selenium derivative from selenide that adds to the 2,3-double bond of aminoacrylyl-tRNA(Sec) generated by selenocysteine synthase to form selenocysteyl-tRNA(Sec). This selenium derivative also is required for the biosynthesis of 2-selenouridine in tRNAs from 2-thiouridine residues. Recently, this reactive selenium derivative formed by the SELD protein was shown to be selenophosphate. From the gene sequence SELD is a 37 kDa protein that contains 7 cysteine residues, two of which are located at positions 17 and 19 in the sequence -Gly-Ala-Cys-Gly-Cys-Lys- Ile-. Inactivation of the enzyme by alkylation with iodoacetamide indicated that at least one cysteine residue in the protein is essential for enzyme activity. To test the possibility that the Cys17 and/or Cys19 residue might be essential these were changed to serine residues by site- specific mutagenesis. The biological activities of the wild type and mutant proteins were studied using E. coli MB08 (selD-) transformed with plasmids containing the selD genes. The plasmid containing the Cys17 mutated gene failed to complement MB08 whereas the Cys19 mutated gene was indistinguishable from wild type. The mutant proteins, like the wild type enzyme, bound to an ATP-agarose matrix showing that their affinities for ATP were unimpaired. Selenide-dependent formation of AMP from ATP was abolished by mutation of Cys17, but the Cys19 mutation had no effect on the ability of the enzyme to catalyze the reaction. These results indicate that Cys17 has an essential role in the catalytic process that leads to the formation of selenophosphate from ATP and selenide.